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Abstract

Background

Long interspersed element type one (L1) actively modifies the human genome by inserting
new copies of itself. This process, termed retrotransposition, requires the formation
of an L1 ribonucleoprotein (RNP) complex, which must enter the nucleus before retrotransposition
can proceed. Thus, the nuclear import of L1 RNP presents an opportunity for cells
to regulate L1 retrotransposition post-translationally. The effect of cell division
on L1 retrotransposition has been investigated by two previous studies, which observed
varied degrees of inhibition in retrotransposition when primary cell strains or cancer
cell lines were experimentally arrested in different stages of the cell cycle. However,
seemingly divergent conclusions were reached. The role of cell division on retrotransposition
remains highly debated.

Findings

To monitor both L1 expression and retrotransposition quantitatively, we developed
a stable dual-luciferase L1 reporter cell line, in which a bi-directional tetracycline-inducible
promoter drives the expression of both a firefly luciferase-tagged L1 element and
a Renilla luciferase, the latter indicative of the level of promoter induction. We
observed an additional 10-fold reduction in retrotransposition in cell-cycle arrested
cells even after retrotransposition had been normalized to Renilla luciferase or L1
ORF1 protein levels. In synchronized cells, cells undergoing two mitoses showed 2.6-fold
higher retrotransposition than those undergoing one mitosis although L1 expression
was induced for the same amount of time.

Conclusions

Our data provide additional support for an important role of cell division in retrotransposition
and argue that restricting the accessibility of L1 RNP to nuclear DNA could be a post-translational
regulatory mechanism for retrotransposition.

Keywords:

Findings

Long interspersed elements type one (LINE-1; L1), the only active autonomous transposable
element in the human genome, have played a major role in human genome evolution and
are also responsible for an increasing number of sporadic human genetic diseases
[1-3]. To make new copies, a source L1 element must successfully navigate through every
stage of the retrotransposition process (that is, transcription, translation, and
target-primed reverse transcription). An essential intermediate step is the formation
of an L1 ribonucleoprotein (RNP) complex between L1 mRNA and proteins
[4-6]. L1 RNP must enter the nucleus before a new copy is made via target-primed reverse
transcription
[7]. Therefore, the nuclear import of L1 RNP presents an opportunity for cells to regulate
L1 retrotransposition post-translationally. As nuclear import can occur passively
when nuclear envelope breaks down during cell division, the efficiency of retrotransposition
is predicted to be higher in actively dividing cells. Indeed, the effect of cell division
has been investigated by two previous studies, which compared the level of L1 retrotransposition
in cell-cycle arrested primary cell strains and cancer cell lines
[8,9]. Although both observed varied degrees of inhibition in retrotransposition when cells
were experimentally arrested in different stages of the cell cycle, one study concluded
that cell division was required for retrotransposition and the other determined that
L1 retrotransposition could occur in non-dividing cells (Table
1). Hence, the role of cell division on retrotransposition remains highly debated to
date.

Development of a stable HeLa Tet-ORFeus cell line

To investigate the effect of cell-cycle arrest on L1 retrotransposition, we wished
to establish an assay system that meets the following criteria: (1) It must be a stable
cell line with an integrated L1 reporter. Having an integrated L1 reporter eliminates
variation in transfection efficiency that is inherent in transient assays. However,
this requirement necessitates the use of an inducible promoter because, otherwise,
L1 insertions will accumulate while the cell line is being established. (2) The promoter
activity (that is, transcription) can be conveniently monitored in parallel to L1
retrotransposition. (3) Both the promoter activity and L1 retrotransposition can be
measured with high sensitivity and in a wide dynamic range. Accordingly, we designed
an inducible dual-luciferase L1 assay vector, pYX056 (Figure
1A; detailed in Additional file
1). The design combined a gene regulation and a gene delivery system. First, the Tet-Off
Advanced Inducible Gene Expression System allows stringent control of L1 expression.
The bi-directional PTight inducible promoter drives expression of Renilla luciferase (Rluc) and a hyperactive
synthetic mouse L1, ORFeus
[10]. The latter is tagged with a firefly luciferase/antisense intron (FlucAI) reporter
cassette
[11]. The bi-directional PTight promoter consists of a modified tetracycline-responsive element flanked by two minimal
CMV promoters. In the presence of doxycycline, the tetracycline-controlled transactivator
advanced (tTA) is complexed with doxycycline and is unable to activate PTight. Upon doxycycline withdrawal, free tTA will bind to PTight and activate both L1 and Rluc transcription. Second, the non-viral two-component
Sleeping Beauty (SB) system enables stable gene delivery. The L1/Rluc bi-directional
expression cassette is flanked by a pair of inverted terminal repeats from SB, and
can be ‘cut and pasted’ into the genome by a hyperactive SB transposase (SB100X)
[12] (Figure
1B). Single cell clones were acquired by limiting dilution method and screened for
the lack of Rluc expression in doxycycline-supplemented medium but high levels of
Rluc expression upon doxycycline withdrawal (Figure
1C; detailed in Additional file
1).

Figure 1.L1 retrotransposition in a HeLa Tet-ORFeus stable cell line. (A) A schematic of the bi-directional inducible L1 construct. The bi-directional tet-responsive
promoter PTight drives the expression of an upstream Rluc cassette and a downstream L1 cassette.
The L1 cassette features coding sequences (that is, ORF1 and ORF2) from the synthetic
L1 ORFeus and an antisense-stranded FlucAI reporter cassette
[11]. In the presence of doxycycline, PTight is inactive. FlucAI can be transcribed from its own SV40 promoter. However, no Fluc
activity is expected because Fluc coding sequence is interrupted by an antisense intron
(sense relative to the L1 cassette). (B) Incorporation of the L1 construct into HeLa-tTA cells. The L1 construct is terminally
flanked by ITRs of the Sleeping Beauty DNA transposon (see panel A). To make a stable cell line, the L1 construct was co-transfected with SB100X into
HeLa-tTA cells. Single cell clones were established through limiting dilution in the
presence of doxycycline. Rluc and Fluc were measured after doxycycline withdrawal.
(C) The rationale of L1 retrotransposition assay with Tet-ORFeus cells. In the absence
of doxycycline, PTight is bound by tTA and activates the transcription of a Rluc mRNA and an L1 pre-mRNA.
The intron is removed from L1 pre-mRNA through splicing. The mature L1 mRNA is reverse
transcribed and integrated into the genome (shown as a 5′ truncated insertion), forming a functional Fluc cassette.

Control of L1 retrotransposition in HeLa Tet-ORFeus cells by doxycycline

To characterize L1 retrotransposition in HeLa Tet-ORFeus cells, we first tested the
dose response by seeding cells in different concentrations of doxycycline. Both Fluc
and Rluc signals were doxycycline dose-dependent (Additional file
2). Significant Fluc signals were first observed after 30 h incubation in doxycycline-free
medium and subsequently increased exponentially to 460-fold above background after
48 h incubation (P <0.01; Figure
2A). The rapid induction of the PTight promoter via doxycycline withdrawal was demonstrated by continued increase of Rluc
signals from three-fold (at 6 h) to 280-fold (at 48 h) above background (P <0.01; Figure
2A). To directly measure L1 expression, we quantified L1 ORF1 protein (ORF1p) by western
blot (Figure
2B). L1 ORF1p signals were first observed at 9 h, peaked at 24 h, and subsequently
maintained for the duration of the experiment (Figure
2B). Induction of Rluc or ORF1p was not observed in HeLa Tet-ORFeus cells cultured
in 100 ng/mL of doxycycline, indicating PTight was completely suppressed. Indeed, cells maintained under 100 ng/mL of doxycycline
showed no accumulation of Fluc-positive cells over 10 passages but could be robustly
induced upon doxycycline withdrawal (Additional file
3). To confirm that Fluc signals were due to retrotransposition, we monitored intron
removal by genomic DNA PCR as previously described
[11]. Consistent with Fluc measurement, the intronless amplicon became most prominent
at 30–48 h although weak amplicons could be observed in earlier time points. As a
control, no intronless band was seen in HeLa Tet-ORFeus cells under 100 ng/mL of doxycycline
(Figure
2C). Similar to the transient dual-luciferase assays
[11], retrotransposition was inhibited by a nucleoside reverse transcriptase inhibitor
in a dose-dependent manner (Additional file
4). The number of L1 insertions was further quantified by a quantitative PCR (qPCR)
method as previously described
[13]. Similar to transient transfection experiments
[11], statistically significant signals were first detected at 24 h (normalized activity
= 5.6%, P <0.01) (Figure
2D). It should be noted that, as opposed to antibiotic or fluorescent protein reporters,
which can be used to track individual retrotransposition events, the HeLa Tet-ORFeus
system measures retrotransposition from a population of cells.

Figure 2.The time course of L1 retrotransposition in HeLa Tet-ORFeus cells. (A) Fluc and Rluc activities from cell lysates. Cells were seeded in 96-well plates
(for luminescence) or 60 mm dishes (for protein and gDNA analyses) in the absence
of doxycycline and harvested at the indicated time points. Error bars represent mean±SE
(n = 6). All readings were compared with the 0 h control (**P <0.01). (B) Time-dependent increase of ORF1p expression. ORF1p and β-actin were detected by
western blot. Murine embryonal carcinoma cells (F9) were used as a positive control
for ORF1p. The parental HeLa-tTA cells and uninduced HeLa Tet-ORFeus cells were used
as negative controls. (C) Confirmation of L1 retrotransposition by end-point PCR. Genomic DNA was amplified
by an intron-flanking primer pair. The presence of a band of 250 bp is diagnostic
for intron removal; the intron-containing donor DNA is amplified as a band of 1150
bp. NTC, no template control. Dox+, gDNA from cells cultured in the presence of doxycycline
for 48 h. Fluc plasmids with or without the intron were used as controls. Molecular
weight was indicated by the 1 kb Plus DNA Ladder (Invitrogen). (D) Quantification of L1 insertions by qPCR. The number of L1 insertions in gDNA was
determined by a TaqMan-based qPCR assay. qPCR signals were normalized by setting signals
from the 48 h time point to 1. The normalized signals from each time point were then
compared with the 0 h time point by two-tailed Student’s t-test. P values are indicated (**P <0.01). Error bars represent mean±SE (n = 3).

Additional file 2: Figure S1.. Dose-dependent induction of L1 retrotransposition in HeLa Tet-ORFeus cells. HeLa Tet-ORFeus
cells were seeded in 96-well plate at 3,000 cells/well and cultured in the presence
of different concentrations of doxycycline. Fluc and Rluc were measured after 48 h
incubation. Error bars represent mean±SE (n=4). At high doses in the range of 6.3 to 100 ng/mL, both Rluc and Fluc showed no
deviation from background readings. Retrotransposition, as indicated by the Fluc signal,
was detected in cells treated with lower doses of doxycycline. In particular, retrotransposition
reached 120-fold above background under 0.8 ng/mL of doxycycline (P <0.001) and 3,600-fold above background in doxycycline-free medium (P <0.001). As expected, the level of retrotransposition was correlated with PTight promoter activity, which was measured by Rluc. At 0.8 ng/mL of doxycycline, Rluc
was induced to five-fold above background (P <0.05); in doxycycline-free medium, Rluc was induced to 620-fold above background
(P <0.001). It should be noted that Fluc signal had increased above background at 1.6
to 3.2 ng/mL concentrations while Rluc activity remained undetectable. This discrepancy
is likely due to the known higher sensitivity of Fluc than Rluc. Thus, our data showed
that L1 retrotransposition efficiency in HeLa Tet-ORFeus cells could be induced by
reducing or eliminating doxycycline from the culture medium. (PDF 57 kb)

Additional file 3: Figure S2.. Induction of L1 retrotransposition in HeLa Tet-ORFeus cells after multiple passages.
HeLa Tet-ORFeus cells were maintained in the presence of 100 ng/mL doxycycline and
passaged in approximately every 3 days. Aliquots of cells from each of the 10 continuous
passages (P0 to P9) were seeded in the presence (Dox+, shown in panel A) or absence
(Dox-, shown in panel B) of 100 ng/mL doxycycline. Fluc and Rluc were measured 48
h after seeding. Note very different scales are used for the two panels. Panel A shows
that Fluc and Rluc signals from uninduced cells are always below 1,000 relative light
units, which represent the assay background and are comparable to readings from empty
wells. Cells from most passages were seeded at the density of 3,000 to 5,000 cells/well
in 96-well plates. The only exception was cells from P2, which were seeded at a much
higher density (40,000 cells/well) in a 96-well plate; this suboptimal seeding density
may explain the much reduced Fluc and Rluc signals in P2 cells in the absence of doxycycline
(panel B). Error bars represent mean±SE (n=4 or 6). In summary, for cells from all passages tested, Fluc and Rluc were completely
inhibited by doxycycline but were consistently induced upon doxycycline withdrawal.
(PDF 67 kb)

Cell-cycle arrest inhibits L1 retrotransposition

To test the effect of cell-cycle arrest in HeLa Tet-ORFeus cells, cells were treated
with three different inhibitors in the absence of doxycycline. Cell-cycle analysis
showed that cells were arrested in S phase by aphidicolin and hydroxyurea and in S+G2/M
phase by thymidine (Figure
3A). The presence or absence of doxycycline had no effect on cell-cycle status (compare
Dox+ with Dox- in Figure
3A). As compared with control cycling cells (that is, Dox-), arrested cells showed
7.6% to 9.4% Rluc expression, indicating PTight was suppressed in the arrested cells (Figure
3B; P <0.001, detailed in Additional file
5: panel A). Indeed, western blot analyses confirmed that, as compared with the Dox-
group, the level of ORF1p was reduced to 6% to 15% in the arrested cells (Figure
3C). If the frequency of retrotransposition was a simple function of L1 expression,
we would expect a proportional reduction of retrotransposition in arrested cells (that
is, approximately 10% of cycling cells). However, the Fluc signal in arrested cells
was at most 0.8% of the Dox- group (Figure
3B; P <0.001, detailed in Additional file
5: panel B), indicating the presence of an additional approximate 10-fold reduction
in retrotransposition that cannot be explained by the decrease in L1 expression. A
potential caveat for these results is that the level of retrotransposition was indirectly
measured by the expression of Fluc from integrated L1 insertions; this may cause an
ascertainment bias between control and treatment groups if the inhibitors affect Fluc
expression. Thus, we directly quantified retrotransposition by qPCR, a method that
is independent of Fluc expression. Results from these qPCR experiments confirmed the
additional reduction in retrotransposition in cell-cycle arrested cells (Figure
3D): in all three treatment groups, the magnitude of decease in qPCR signal was greater
than the fold reduction in L1 expression, regardless whether L1 expression is measured
as Rluc or ORF1p. On the other hand, two out of the three inhibitors displayed an
inhibitory effect on Fluc expression when the Fluc data and qPCR data were compared
(0.09% versus 0.4% for hydroxyurea treated cells and 0.8% versus 3.8% for thymidine treated cells, respectively; compare Figure
3B and
3D). As a result, we compared the correlation between ORF1p and qPCR data. After adjusting
the decrease in ORF1p, qPCR showed additional 8.7-, 27.5-, and four-fold reductions
in retrotransposition in aphidicolin, hydroxyurea, and thymidine treated cells, respectively
(compare Figure
3C and
3D).

Figure 3.Cell-cycle arrests inhibit L1 retrotransposition in HeLa Tet-ORFeus cells. (A) Cell-cycle analysis. HeLa Tet-ORFeus cells were cultured in doxycycline-free medium
(Dox-) or supplemented with 5 μg/mL aphidicolin, 75 μg/mL hydroxyurea, or 2 mM thymidine.
Cells cultured in 100 ng/mL doxycycline were used as control (Dox+). The distribution
of cells in different phases of the cell cycle and their corresponding DNA content
histograms are shown. (B) Normalized Fluc and Rluc activities. HeLa Tet-ORFeus cells were treated as in panel
A for 48 h. Raw luminescence readings were normalized by cell viability first and
then to those from Dox- cells (Flucmean = 425,000 and Rlucmean = 314,000). Error bars represent mean±SE (n=6). Statistical analyses are presented in Additional file
5. (C) The effect of cell-cycle arrest on ORF1p expression. Representative western blots
were shown for ORF1p and β-actin; quantitative data were calculated from three biological
replicates and had been normalized by β-actin. F9 cells were used as a positive control
for ORF1p. The parental HeLa-tTA cells and uninduced HeLa Tet-ORFeus cells (Dox+)
were used as negative controls. (D) Quantification of L1 insertions by qPCR. The number of L1 insertions in gDNA was
determined by a TaqMan-based qPCR assay. qPCR signals were normalized by setting signals
from the Dox- cells to 1 (equivalent to 4.9 copies per cell as estimated from plasmid
DNA dilution series). Error bars represent mean±SE (n=3).

Additional file 5: Figure S4.. Effect of cell-cycle arrests on Rluc and Fluc activities in HeLa Tet-ORFeus cells.
The underlying data are the same as in Figure 3B but Rluc and Fluc data are separately
graphed to highlight the difference among experimental conditions. Raw Rluc (panel
A) and Fluc (panel B) readings are shown underneath the x-axis labels. They were normalized
by cell viability first and then to those from Dox- cells and plotted. Error bars
represent mean±SE (n=6). Pairwise two-tailed Student’s t-test was used to compare Rluc or Fluc signals between treatment groups; resulting
P values are indicated (*P <0.05, **P <0.01, ***P <0.001). (PDF 91 kb)

L1 retrotransposition in synchronized HeLa Tet-ORFeus cells

To exclude the possibility that the observed inhibition of L1 retrotransposition is
caused by unknown side effects of inhibitors used, we wished to test the effect of
cell division on L1 retrotransposition in cycling cells. To this end, we synchronized
HeLa Tet-ORFeus cells by double-thymidine block and then allowed cells to enter normal
cell cycling by removing thymidine from the culture medium (Figure
4A). According to cell-cycle analysis, these cells would complete two full cell cycles
in 44 h (Figure
4A; Additional file
6). We compared retrotransposition under two experimental conditions (b and d in Figure
4A). L1 transcription was induced for the same amount of time in both conditions (that
is, 37 h). However, the withdrawal of doxycycline was timed so that L1 expression
was activated at different cell-cycle phases. As a result, when L1 expression was
induced, cells in experiment b would undergo G2/M phase once whereas cells in experiment
d would undergo G2/M phase twice (Figure
4A). After 37 h induction, dual-luciferase readouts were taken from both conditions.
Both showed similar levels of promoter activities (Figure
4B) but the level of retrotransposition was 2.6-fold higher in experiment d than in
experiment b (P <0.05; Figure
4C). In control experiments, we demonstrated that the difference in assay duration
did not alter the assay background (Figure
4A to C; conditions a and c, where cells were released from the double-thymidine block
but remained in doxycycline-supplemented medium). Thus, these data from synchronized
cells further support the conclusion that cell division promotes L1 retrotransposition,
and thus is a potential means of regulating L1 activity.

Additional file 6: Figure S5.. Cell-cycle progression after HeLa Tet-ORFeus cells released from double-thymidine
block. HeLa Tet-ORFeus cells were synchronized at G1/S phase and subsequently allowed
to cycle by incubating in complete medium in the absence of thymidine and doxycycline.
The time of release from thymidine block was designated as time 0. Cells were collected
every 4 h and subjected to cell-cycle analysis. The distribution of cell-cycle phases
(G1, S, and G2/M) was plotted over time. The first column ‘C’ denotes a control population
of unsynchronized cells. Note cells progressed through the first full cycle (from
S, G2/M, G1 to the next S) within the first 20 h relatively synchronously but the
second cycle was not as synchronous as the first cycle. (PDF 75 kb)

In summary, our data provide additional support for an important role of cell division
in L1 retrotransposition and argue that restricting the accessibility of L1 RNP to
nuclear DNA could be a post-translational regulatory mechanism for retrotransposition
(Table
1). As compared with the two previous studies
[8,9], our experimental approach has several advantages for assessing the role of cell
division in retrotransposition. First, the dual-luciferase system provided an efficient
means of simultaneous quantification of both L1 expression and L1 retrotransposition.
Second, the use of an inducible, integrated reporter not only allowed us to avoid
variation in gene transfer efficiency between experimental conditions but also to
better resemble the replication cycle of endogenous L1 elements, which express from
chromosomal rather than episomal DNA. Indeed, it allowed us to separate two layers
of regulation in cell-cycle arrested cells: one layer is at the transcriptional level,
which was highlighted by Shi et al.
[9]; the other layer is downstream and independent of L1 transcription, as indicated
by both Rluc signals and ORF1p levels (discussed below). Lastly, our inducible system
permitted the comparison of retrotransposition in synchronized cell populations where
the major difference was the number of mitoses completed.

Integrating our data and those of previous studies
[8,9], we propose that active cell division promotes retrotransposition. All three studies
showed strong inhibition of retrotransposition when cells were arrested. Shi et al.
[9] analyzed L1 RNA levels in their assay system and attributed the inhibitory effect
on retrotransposition largely to reduced L1 transcription (Table
1). The assay systems used by this study and Kubo et al.
[8] enabled retrotransposition at larger dynamic ranges, permitting the evaluation of
additional layers of regulation. In this study, after Fluc signals were normalized
to the co-expressed Rluc, we observed an additional 10-fold reduction in retrotransposition
in cell-cycle arrested HeLa Tet-ORFeus cells and a 2.6-fold reduction in synchronized
cells undergoing one fewer round of cell division. Thus, after factoring in the effect
of drug treatment on L1 expression, our data support an important role of cell division
in promoting efficient L1 retrotransposition in a manner independent of L1 expression.
It is noteworthy that, even when the variable infection rate was not taken into consideration,
Kubo et al.
[8] found a three-fold reduction of retrotransposition in G1/S arrested cells in addition
to a 16-fold reduction of retrotransposition in G0 arrested Gli36 cells (Table
1). On the other hand, both Kubo et al.
[8] and the current study showed substantial retrotransposition in cell-cycle arrested
cells (for two of the three inhibitors tested, we observed statistically significant
Fluc signals at approximately 10-fold above the assay background). Currently, we cannot
exclude the possibility that the residual retrotransposition observed in arrested
cell populations in both studies originates from a minor population of cycling cells.
An alternative explanation for such residual retrotransposition is that L1 retrotransposition
may also be facilitated by a yet uncharacterized active nuclear import mechanism (Table
1). Indeed, the control experiments performed by Kubo et al.
[8] in G1/S arrested cells, showing differential transduction by retroviral and lentiviral
vectors, support the presence of an active nuclear import mechanism for L1 retrotransposition.
It is noteworthy that some non-LTR (long terminal repeat) retrotransposons have evolved
active nuclear import strategies for their propagation in respective host species.
A precedent of active nuclear import has been reported for the telomeric repeat-specific
SART1 retrotransposon from Bombyx mori: its ORF1p contains functional nuclear localization signals (NLSs), which are required
for active retrotransposition
[14]. Thus far, no NLS has been reported in mammalian L1 proteins although both ORF1 and
ORF2 proteins have highly basic regions, which is a common feature of nuclear localized
proteins. Alternatively, it is possible active nuclear import is mediated by other
host-derived components of L1 RNP. Recently, two poly(A) binding proteins, PABPN1
and PABPC1, were found to be associated with L1 RNP
[15]. Of particular interest, PABC1 was found to be critical for RNP formation; as it
can shuttle between the cytoplasm and the nucleus, it would be interesting to determine
whether PABPC1 mediates RNP nuclear import
[15].

A caveat shared by all three studies is that the role of cell division in retrotransposition
was mainly assessed in cancerous cell lines (Table
1; but note Shi et al.
[9] also tested normal human fetal lung fibroblasts). Additionally, two of these studies
(
[8] and the current study) used non-native promoters to drive L1 expression (Table
1), which precludes the study of the endogenous transcriptional regulation of L1 with
these systems. Nevertheless, a unifying view from these and other studies of L1 variants,
mutations, and host factors (
[15-19] and citations therein) is that retrotransposition is not a simple function of L1
expression. By extension, the level of L1 expression cannot be equated with the frequency
of retrotransposition and the evaluation of retrotransposition should take into consideration
the cell-cycle status. For example, in normal individuals, endogenous L1 expression
has only been confirmed at protein level in testicular and ovarian tissues (
[20-22]; reviewed in
[23]). It is noteworthy that L1 ORF1p is detected in two distinct stages of male germ
cell development, namely, in gonocytes (embryonic stage) and in meiotic/post-meiotic
germ cells (prepuberal and through adulthood)
[20-22]. However, during both stages cell division is limited: gonocytes are mitotically
arrested in G0 phase
[24] while spermatocytes only divide twice before becoming haploid spermatids. Similarly,
in the female germline, L1 ORF1p is detected during the meiotic prophase I in embryonic
oocytes
[21], which are subsequently arrested in the diplotene stage of the meiotic prophase I
and do not divide until puberty
[25]. Therefore, both male and female germline development may have been programmed in
a way that restricts excessive retrotransposition by avoiding frequent nuclear membrane
breakdown when L1 is expressed. Thus, to understand the developmental timing of retrotransposition,
it is imperative to measure the level of retrotransposition directly.

Competing interests

The authors declare that they have no competing financial interests.

Authors’ contributions

YX performed the studies and drafted the manuscript. LM, ZIv, ZIz, and SM contributed
reagents and provided scientific consultation. WA directed the studies and finalized
the manuscript. All authors read and approved the final manuscript.

Acknowledgements

We thank all the An lab members for helpful discussions. This work was funded by the
College of Veterinary Medicine of Washington State University, which did not have
any role in the study design, data collection, analysis and interpretation of data,
or in the writing of the article and the decision to submit it for publication.